Effective interstitial transport of particles is necessary for injected drug/diagnostic agents to reach the intended target; however, quantitative methods to estimate such transport parameters are lacking. In this study we develop an in vivo model for evaluating interstitial convection of injected macromolecules and nanoparticles. Fluorescently labeled macromolecules and particles are co-infused with a reference solute at constant infusion pressure intradermally into the mouse tail tip, and their relative convection coefficients are determined from spatial and temporal interstitial concentration profiles. Quantifying relative solute velocity with a co-infused reference solute eliminates the need to estimate interstitial fluid velocity profiles, greatly reducing experimental variability. To demonstrate sensitivity and usefulness of this model, we compare the effects of size (dextrans of 3, 40, 71, 2000 kDa and 40 nm diameter particles), shape (linear dextran 71 kDa vs. 69 kDa globular protein albumin), and charge (anionic vs. neutral dextran 3 kDa) on interstitial convection. We find significant differences in interstitial transport rates between each of these molecules and confirm expected transport phenomena, testifying to sensitivity of the model in comparing solutes of different size, shape and charge. Our data show that size exclusion (within a specific size range) dominates molecular convection, while mechanical hindrance slows larger molecules and nanoparticles; proteins convect slower than linear molecules of equal molecular weight, and negative surface charges increase convection through matrix repulsion. Our in vivo model is demonstrated to be a sensitive and reliable tool for evaluating and optimizing potential drug/diagnostic vehicles that utilize interstitial and lymphatic delivery routes.